Nature of Large Vesicle Exocytosis in Pancreatic β-cells: Release of ATP and GABA
Sammanfattning: Several high resolution electrophysiological techniques such as measurements of cell membrane capacitance and amperometry are used to detect exocytotic events. A novel method was developed in this study based on the expression of P2X2 receptor channel in secreting cells. The presence of ATP sensitive channel on the cell membrane permitted the electrical detection of minute (amoles) quantities of ATP and thus created a possibility to measure ATP released from a single insulin granule. The release of peptides and low molecular weight substances was explored by combining the detection of exocytotic events by P2X2 receptor method with imaging of secretory granules. It was found that in rat insulinoma cells these substances were released independently and with different time course: nucleotides - 280 ms and granular peptide marker IAPP - 2.2 s after the membrane depolarization. In 72% of all exocytotic events nucleotide release was not followed by the discharge of peptide cargo. The chief inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is also found in pancreatic β-cells though its role in coordinating processes in the pancreas is unknown. Sub-cellular localization has not been fully resolved too. Electrical measurements of ATP, GABA and 5-HT release in rat pancreatic β-cells allowed us to demonstrate that at least 75% of GABA release events were attributable to the exocytosis of large dense core vesicles in these cells and its selective release was regulated by the size of the fusion pore. The combination of the cell capacitance measurements, the electrical measurements of ATP release and amperometric detection of 5-HT release demonstrated that in rat pancreatic β-cells ATP is stored and released by large dense core vesicles. The contribution of small synaptic like vesicles is below the detection levels of methods used. The possibility of multi-vesicular exocytosis in pancreatic β-cells was explored by the cell-attached capacitance measurements, the total internal reflection microscopy and by electrical ATP release measurements. It was found that global increase in [Ca2+]i promotes the formation of complexes of interconnected granules within cell cytoplasm that subsequently undergo exocytosis as one unit. This may lead to exocytosis of up to 15 granules simultaneously (compound exocytosis).
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